1. Field of the Invention
The present invention relates to enzyme electrodes. More particularly, it relates to electrodes for amperometrically measuring concentrations in an electrolyte of those substances which can be the substrate for oxidases, such as glucose, galactose and the like, or of those substances which can be the substrate for dehydrogenases, such as lactic acid, alcohol, glycerol and the like.
2. Description of the Prior Art
In recent years, increasing attention has been paid to oxidoreductase immobilized electrodes (hereinafter, referred to as enzyme electrodes) (reference is made to T. Ikeda, S. Ando, and M. Senda, Bull. Chem. Soc. Jpn., 54, 2189 (1981)). These electrodes behave as a substitute for a chemical electron transporter in enzyme reaction, and it has been suggested that they could be utilized in such novel applications as enzyme electrodes, detectors in flowing systems biochemical fuel cells and enzyme reactors (reference is made to S. Ando, T. Ikeda, T. Kakutani, and M. Senda, Rev. Polarogr. (Kyoto), 26, 19(1980): abstracts of papers presented at the Annual Meeting on Polarography, October 1980, Fukuoka).
Such enzyme electrodes are of the form directly utilizing current (electric current and voltage related to enzyme reaction, being different from those electrodes of the form indirectly estimating the amount of substrate contributing to enzyme reaction by measuring the amount of a product produced by the enzyme reaction, for example, the amount of hydrogen peroxide generated, with the so-called oxygen electrode or platinum electrode whose sensitive surface is coated with an enzyme-immobilized film.
In this regard, the inventors of the present invention have previously found that glucose oxidase-immobilized electrodes using graphite as a carrier can function as a bioelectrocatalysis electrode for electro-oxidation of glucose in the presence of an electron transfer mediator such as p-benzoquinone in solution (reference is made to T. Ikeda, I. Katasho, M. Kamei, and M. Senda, Agric. Biol. Chem., 48, (8) (1984)).
In such oxidase electrodes, however, there were problems on practical use. For example, (a) it was required to add to the solution to be measured a substance working as the electron transfer mediator participating in the enzyme reaction, at every time of measurement, (b) since the amount of such substance capable of contributing to the reaction was regulated by its concentration in the solution and this varied by concentration polarization during electrolysis, the substance could not be supplied to the reaction system in a high and constant concentration and accordingly the response speed and the sensitivity were insufficient, and (c) the influence on the substance of these substances coexisting in the solution to be measured, pH of the solution, oxygen contained in the solution, etc., as well as that of light, could not be disregarded.
On the other hand, it has been also suggested to form an oxidation-reduction system by utilizing immobilized dehydrogenase in combination with nicotinamide adenine dinucleotide (NAD) as an electron transfer mediator and use the system for an electrode reaction. Moreover, in order to carry out the measurement without adding the NAD to the solution to be measured at every time of measurement as described above, it has been tried to immobilize NAD together with enzyme. As representatives of such trials 1 electrodes prepared by trapping a NAD-high molecular weight compound obtained by chemically binding NAD with a high molecular weight compound, such as agarose or dextran, on the inside surface of a suitable substrate-permeable film together with enzyme and fixing the film to a platinum or graphite electrode, .circle.2 electrodes prepared by chemically binding NAD directly with a substrate-permeable film, trapping enzyme on the inside surface of the film and simultaneously fixing the film to a platinum or graphite electrode, .circle.3 electrodes prepared by applying chemical treatment to a substratepermeable film to make it hardly permeable for NAD, trapping enzyme on the inside surface of the film and fixing the film to a platinum or graphite electrode, and the like have been suggested.
However, the above-described enzyme electrodes .circle.1 and .circle.2 involved the problem that the enzyme reactivity of NAD itself was largely decreased by incorporation of NAD into a high molecular weight compound, and the electrodes .circle.3 involved the problem that leakage of NAD could not be prevented perfectly and the enzyme reactivity lowered as time passed.
The present invention is one which is made in view of various problems as mentioned above, and one of its purposes is to provide enzyme electrodes capable of maintaining a high reactivity of immobilized enzyme for a long period of time without adding any electron transfer mediator to the solution to be measured.